1. Field of the Invention
The present invention relates to a fermentation method for producing hyaluronic acid. More specifically, it relates to a fermentation method for producing streptolysin-free hyaluronic acid by cultivating (or incubating) a microorganism belonging to genus Streptococcus.
2. Description of the Related Art
Hitherto, Hyaluronic acid has been isolated from the hyaloid liquid of bovine eyes, cockscombs, umbilici, and articulation liquid of chickens and is known to be used as a substance which maintains a jelly state by bonding with proteins or the other mucopolysaccharides and water to thereby act as a lubricant, to prevent the invasion of bacteria, and retain water. However, the above method is disadvantageous in that the cost of obtaining the hyaluronic acid is too high.
It has been reported in, for example, B. Holmstrom, Appl. Microbial, 15(6), 1409-1413, 1967, J. B. Woolcock, J. Gen. Microbial, 85, 372-375, 1974, and E. Kjem, Acta Pathol. Microbial. Scand, 84(3), 162-164, 1976 that hyaluronic acid can be produced relatively cheaply by utilizing bacteria strains belonging to the genus Streptococcus such as Streptococcus pyogenes, Streptococcus equi, Streptococcus equisimilis, Streptococcus dysgalactiae, and Streptococcus zooepidemicus. However, according to Bergey's Manual of Determinative Bacteriology (8th edition, 1974), all of the above-mentioned strains belong to biogenic groups of genus Streptococcus, such as lactic acid and A or C type bacteria of a Lancefield serum group. That is, these strains are bacteria generally known as hemolytic streptococci, and are known to exhibit .beta.-hemolysis by soluble hemolysin, i.e., streptolysin. Although correlationship between the streptolysin producing capability and the pathogenicity is not clearly understood, the hyaluronic acid product is likely to be unpreferably contaminated with the above-mentioned proteins when hyaluronic acid is industrially produced from the cultivation of the above-mentioned strains. Especially, when wishing to incorporate hyaluronic acid into medical compositions for external use or into cosmetic compositions, it is preferable that streptolysin be completely removed from the hyaluronic acid product since these compositions are applied to the skin.
When subculturing of the above-mentioned strains is continued, not only is the hemolysis gradually decreased but also the hyaluronic acid producing capability is decreased, and the mucoid colonies disappear. Furthermore, the hemolysis of the strains still remains after repeated subculturing. Consequently, it is very difficult or almost impossible to effectively produce hyaluronic acid not contaminated by streptolysin.